1. Field of the Invention
The present invention relates to a composition for hepatoprotection and treatment of liver diseases comprising a dihydroxyphenyl derivative represented by the following formula (1) which has an excellent hepatoprotective and therapeutic activity for liver diseases: 
in which
A and B independently of one another represent hydrogen, or together represent methylene group,
D represents hydrogen or lower alkoxy,
E represents acetylthioacetyl, or represents the following substituent (a-1) or (a-2): 
wherein
 represents single or double bond,
R1 represents hydrogen, lower alkyl or N-acetylmethylaminomethyl,
R2 represents hydrogen, or represents lower alkyl which is optionally substituted by hydroxycarbonyl, phenyl or 5- or 6-membered heteroaryl containing one or more hetero atoms selected from a group consisting of nitrogen and sulfur, wherein the heteroaryl can be substituted by lower alkyl,
R3 represents hydrogen, hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbamoyl or lower alkenylcarbamoyl, or represents lower alkyl which is substituted by hydroxy, aryl(lower)alkoxy or 5- or 6-membered heteroaryl(lower)alkoxy containing nitrogen as the hetero atom,
R4 represents hydrogen, or represents lower alkanoyl which is optionally substituted by halogen,
provided that R4 does not exist when double bond is linked to the nitrogen atom in substituent (a-2).
The present invention also relates to a novel dihydroxyphenyl derivative having an excellent protective and therapeutic acitivity for liver which is left after known compounds are removed from the compound of formula (1) above, and to a process for preparing the same.
2. Description of the Prior Art
The liver has been known as an important organ wherein various metabolic activities are carried out. Acute or chronic lesions can be developed by a variety of factors including noxious materials such as virus, chemicals, etc., and undernourishment, which may cause liver injuries such as fatty liver, hepatitis, jaundice, cirrhotic liver, hepatic sclerosis, and liver cancer, etc. Recently, as the therapeutic agents for liver diseases, silymarin(see, Biotech, Therapeutics, 1993, 4, 263-270), malotilate(see, Japan, J. Exp. Med., 1986, 56, 235-245; Biochem, Biophy. Res. Comm., 1994, 200, 1414, 1994), DDB(see, Biochem. Biophy. Res. Comm., 1981, 103, 1131-1137), flumecinol(see, U.S. Pat. No. 4,039,589), etc. were reported, and yet which also have been proved to have demerits of their own. That is, silymarin has a low bioavailability and medicinal effect; malotilate exhibits a hematotoxicity and low bioavailability in oral administration and further it causes undesirable side effects due to its metabolite; flumecinol can be restrictively used to an infant; and DDB has a low bioavailability when administered orally. In addition, a dietary cure, symptomatic treatment, and medical therapies using steroids, immune-related agent, etc. are known, but the efficacy thereof as a therapeutic agent is immaterial. Further, the present inventors have identified the therapeutic effect of novel genipin derivatives to hepatitis.
While, the following compounds of formulas (2) to (4) may be mentioned as reference compounds having a similar structure to that of the compound of formula (1) according to the present invention and also similar use. First, the thiazofuran of formula (2) as a thiazole derivative acts as an inhibitor for purine biosynthesis, by which it exhibits a therapeutic effect to myelocytic leukemia. Therefore, it is now in a third clinical test by ICN company. 
Also, the thiazoline derivative of the following formula (3) has been reported to have parasiticidal and fungicidal effects(see, Pharm. Acta. Helv. 1991, 66(8), 237-40), and the pidotimod of the following formula (4) which is a thiazolidine derivative has been marketed by Poli Industria Chimica since 1993 as an immuno-regulatory agent through the mechanism of PNP(purine nucleoside phosphorylase) inhibition. 
in which
R represents phenyl, substituted phenyl, 2-furyl, 1-naphthyl or 4-pyridyl. 
Under such a technical background as mentioned above, the present inventors have extensively studied to develop novel compounds which can be effectively used in the hepatoprotection and treatment of liver diseases. As a result, we have succeeded to identify that the compound of formula (1) according to the present invention exhibits a potent hepatoprotective and therapeutic activity for liver diseases, and further part of the compound of formula (1) is novel.
Therefore, it is an object of the present invention to provide a pharmaceutical composition for the hepatoprotection and treatment of liver diseases comprising as an active ingredient a dihydroxyphenyl derivative of formula (1), as defined below, pharmaceutically acceptable acid addition salt or stereochemical isomer thereof together with a pharmaceutically acceptable inert carrier. 
in which
A and B independently of one another represent hydrogen, or together represent methylene group,
D represents hydrogen or lower alkoxy,
E represents acetylthioacetyl, or represents the following substituent (a-1) or (a-2): 
wherein
 represents single or double bond,
R1 represents hydrogen, lower alkyl or N-acetylmethylaminomethyl,
R2 represents hydrogen, or represents lower alkyl which is optionally substituted by hydroxycarbonyl, phenyl or 5- or 6-membered heteroaryl containing one or more hetero atoms selected from a group consisting of nitrogen and sulfur, wherein the heteroaryl can be substituted by lower alkyl,
R3 represents hydrogen, hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbamoyl or lower alkenylcarbamoyl, or represents lower alkyl which is substituted by hydroxy, aryl(lower)alkoxy or 5- or 6-membered heteroaryl(lower)alkoxy containing nitrogen as the hetero atom,
R4 represents hydrogen, or represents lower alkanoyl which is optionally substituted by halogen,
provided that R4 does not exist when double bond is linked to the nitrogen atom in substituent (a-2).
Part of the compound of formula (1) which is left after known compounds are removed therefrom is novel, and therefore it is another object of the present invention to provide such a novel dihydroxyphenyl derivative and processes for the preparation thereof.
The novel dihydroxyphenyl derivative according to the present invention is the compound of formula (1) wherein
A and B independently of one another represent hydrogen, or together represent methylene group,
D represents hydrogen or lower alkoxy,
E represents acetylthioacetyl, or represents the following substituent (a-1) or (a-2): 
wherein
 represents single or double bond,
R1 represents hydrogen, lower alkyl or N-acetylmethylaminomethyl,
R2 represents hydrogen, or represents lower alkyl which is optionally substituted by hydroxycarbonyl, phenyl or 5- or 6-membered heteroaryl containing one or more hetero atoms selected from a group consisting of nitrogen and sulfur, wherein the heteroaryl can be substituted by lower alkyl,
R3 represents hydrogen, hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbamoyl or lower alkenylcarbamoyl, or represents lower alkyl which is substituted by hydroxy, aryl(lower)alkoxy or 5- or 6-membered heteroaryl(lower)alkoxy containing nitrogen as the hetero atom,
R4 represents hydrogen, or represents lower alkanoyl which is optionally substituted by halogen,
provided that
i) R1 and R2 are not hydrogen or methyl when D is hydrogen and E is substituent (a-1),
ii) R3 is not hydrogen in the thiazoline ring of (a-2) when D is hydrogen and E is substituent (a-2), and
iii) R4 does not exist when double bond is linked to the nitrogen atom in substituent (a-2).
More preferred composition according to the present invention comprises a dihydroxyphenyl derivative represented by the following formula (1): 
in which
A and B both represent hydrogen, or together represent a methylene group,
D represents hydrogen or C1-C4-alkoxy,
E represents the following substituent (a-1) 
wherein
R1 represents hydrogen, C1-C4-alkyl or N-acetylmethylaminomethyl,
R2 represents hydrogen, or represents C1-C4-alkyl which is optionally substituted by hydroxycarbonyl, phenyl or 5- or 6-membered heteroaryl containing one or more hetero atoms selected from a group consisting of nitrogen and sulfur, wherein the heteroaryl can be substituted by C1-C4-alkyl,
provided that when A and B together form a methylene group, D is hydrogen, R1 is hydrogen, and R2 is not hydroxycarbonylethyl; and
provided that when A, B, D and R1 independently are hydrogen, R2 is not hydrogen.
Among the novel compound of formula (1) according to the present invention, the preferred compounds include those wherein D represents hydrogen or methoxy, E represents substituent 
wherein R1 represents hydrogen or methyl, and R2 represents hydrogen, methyl, t-butyl, hydroxycarbonylmethyl, benzyl, 2-pyridylmethyl or 4-methylthiazol-5-ylethyl.
Also preferred compounds of formula (1) include those wherein D represents hydrogen or methoxy, E represents subsituent (a-2), wherein represents single or double bond, R3 represents hydrogen, hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbamoyl, lower alkenylcarbamoyl, or represents lower alkyl substituted by hydroxy, benzyloxy or 2-pyridylmethoxy, and R4 represents hydrogen, or lower alkanoyl which is optionally substituted by halogen. Particularly preferred compounds among these compounds are those wherein D represents hydrogen, E represents substituent (a-2), wherein R3 represents hydrogen, hydroxycarbonyl, ethoxycarbonyl, methyl-carbamoyl, allylcarbamoyl, or methyl substituted by hydroxy, benzyloxy or 2-pyridylmethoxy, and R4 represents hydrogen, acetyl or chloroacetyl. Most preferred one is the compound wherein R3 and R4 are both hydrogen on the thiazole ring.
Also, preferred compounds of formula (1) include those wherein D represents hydrogen or methoxy, and E represents acetylthioacetyl.
Typical examples of the compound of formula (1) are represented in the following table 1. Among them, all the compounds except for Compound Nos. 1 to 5 are novel.
The compound of formula (1) according to the present invention can form a pharmaceutically acceptable salt. Such salt includes a salt with pharmaceutically acceptable acids such as asparagic acid, gluconic acid, hydrochloric acid, p-toluenesulfonic acid or citric acid, etc., a salt with bases such as pyridine or ammonia, etc., and a salt with acids or bases which are generally known and conventionally used in the technical field to which the compound of formula (1) pertains. These pharmaceutically acceptable salts can be prepared according to a conventional conversion method.
In the compound of formula (1) wherein E represents substituent (a-2), the two carbon atoms linked to the nitrogen atom on the 5-membered ring can be asymmetric, and thus the compound of formula (1) can exist as a pure stereoisomer such as enantiomer of R or S, diastereomer, etc., or a mixture thereof including racemate. Therefore, the present invention also includes each of these stereoisomers and their mixtures.
The compound of formula (1) of the present invention can be prepared according to the methods described below. However, it should be understood that the process for preparing the compound of formula (1) is not limited to those explained below since the compound can be easily prepared by optionally combining the various methods disclosed in prior arts, and such a combination may be conventionally carried out by a person having ordinary skill in the art.
According to the present invention, the novel compound of formula (1) can be prepared by processes characterized in that
(a) a compound represented by the following formula (5): 
wherein A, B, D and R1 are defined as previously described, is reacted with a compound represented by the following formula (6):
xe2x80x83xe2x80x83(6) 
wherein R2 is defined as previously described, in a solvent to produce a compound represented by the following formula (1a): 
wherein A, B, D, R1 and R2 are defined as previously described (see, Reaction Scheme 1);
(b) a compound represented by the following formula (7): 
wherein A, B, D and R1 are defined as previously described, is reacted with a compound represented by the following formula (8):
X-R2xe2x80x83xe2x80x83(8) 
wherein R2 is defined as previously described and X represents reactive leaving group, to produce the compound of formula (1a) (see, Reaction Scheme 2);
(c) piperonal is reacted with 1-cysteine ethylester hydrochloride in the presence of pyridine to produce a compound represented by the following formula (9): 
which is then reacted with lower alkanoyl halide or anhydride optionally substituted by halogen to produce a compound represented by the following formula (1b): 
wherein R4xe2x80x2 represents lower alkanoyl optionally substituted by halogen (see, Reaction Scheme 3);
(d) an ethylimidate compound represented by the following formula (10): 
is reacted with mercaptoethylamine hydrochloride or L-cysteine (lower) alkylester hydrochloride to produce a compound represented by the following formula (1c) having a thiazoline ring: 
wherein R3xe2x80x2 represents hydrogen or lower alkoxycarbonyl (see, Reaction Scheme 4);
(e) the compound of formula (1c) is oxidized using an oxidant to produce a compound represented by the following formula (1d) having a thiazole ring: 
wherein R3xe2x80x2 is defined as previously described (see, Reaction Scheme 6);
(f) a compound represented by the following formula (11): 
wherein  is defined as previously described, is reacted with lithium hydroxide, lower alkylamine or lower alkenylamine to produce a compound represented by the following formula (1e): 
wherein R3xe2x80x3 represents hydroxycarbonyl, lower alkylcarbamoyl or lower alkenylcarbamoyl (see, Reaction Scheme 7);
(g) the compound of formula (11) is reduced to produce a compound represented by the following formula (12): 
wherein  is defined as previously described, which is then reacted with aryl(or heteroaryl)(lower)alkyl halide or aryl(or heteroaryl)(lower)-alkylmethylsulfonyloxy to produce a compound represented by the following formula (1f): 
wherein R3xe2x80x3xe2x80x2 represents lower alkyl substituted by aryl(lower)alkoxy or 5- or 6-membered heteroaryl(lower)alkoxy containing nitrogen as the hetero atom (see, Reaction Scheme 8); or
(h) 3xe2x80x2,4xe2x80x2-methylenedioxyacetophenone is reacted with bromine to produce a compound represented by the following formula (13): 
which is then reacted with thiolacetic acid to produce a compound represented by the following formula (1g): 
(see, Reaction Scheme 9).
The above processes (a) to (h) will be explained more specifically below together with the Reaction Schemes. 
The compound of formula (5) used as a starting material in the process variant (a) according to Reaction Scheme 1 above is disclosed in Indian J. Chem., 6(6), 337-8, 1968, and can be prepared according to the procedure described therein. Any inert solvent which does not adversely affect to the reaction, preferably one or more selected from a group consisting of water, methanol, ethanol and isopropyl alcohol, particularly preferably a solvent mixture of methanol and water(10:1, v/v) can be used in process (a). In addition, inorganic bases including sodium hydroxide, potassium hydroxide, etc., or organic bases including triethylamine, etc. can be used as a reaction aid. This reaction is carried out for 2 to 5 hours at room temperature. 
The compound of formula (7) used as a starting material in the process variant (b) according to Reaction Scheme 2 above may be prepared by oxidizing the corresponding hydroxyimino compound in the presence of sodium hydride. As examples of the reactive leaving group in the compound of formula (8), methanesulfonyloxy group can be mentioned. For example, the hydroxy group of 2-pyridylmethanol may be reacted with methanesulfonylchloride in a solvent such as methylene chloride, chloroform, etc. in the presence of a base such as triethylamine, diisopropylethylamine, etc. at xe2x88x9210 to 0xc2x0 C. to produce the compound of formula (8) wherein X is methanesulfonyloxy and R2 is 2-pyridylmethyl. 
In the process variant (c) according to Reaction Scheme 3 above, piperonal is first reacted with L-cysteine ethylester hydrochloride in the presence of pyridine using a nonpolar solvent such as benzene, toluene, xylene, etc. under room temperature to warming to produce the compound of formula (9) which corresponds to the compound of formula (1) wherein R3 on the thiazolidine ring is ethoxycarbonyl. Subsequently, the compound of formula (9) thus obtained is reacted with lower alkanoyl halide or anhydride which is optionally substituted by halogen, such as for example, acetyl chloride, acetic anhydride or chloroacetyl chloride in the presence of an organic base such as pyridine or triethylamine and catalytic amount of dimethylaminopyridine to produce the compound of formula (1b) wherein R4xe2x80x2is lower alkanoyl optionally substituted by halogen. 
The ethylimidate compound used as a starting material in the process variant (d) according to Reaction Scheme 4 above may be prepared from piperonal. That is, the aldehyde group of piperonal is converted to hydroxyimino group according to the same procedure as Reaction Scheme 1, then the compound thus obtained is reacted with p-toluenesulfonylchloride or methanesulfonylchloride in a solvent such as methylene chloride or chloroform in the presence of an organic base such as triethylamine or diisopropylethylamine at 0xc2x0 C. to room temperature in order to convert the hydroxyimino group to a nitrile group. Then, the nitrile group of the resulting compound is converted to an ethylimidate group by reacting it with ethanol saturated with hydrochloric acid gas for 5 hours to one day at 0xc2x0 C. to room temperature. This procedure can be specifically depicted as the following 
In the process variant (e) according to Reaction Scheme 6 above, the thiazoline ring of the compound of formula (1c) prepared in the above process variant (d) is oxidized to a thiazole ring in the presence of an oxidant in a nonpolar solvent such as benzene, toluene, xylene, etc., or in a solvent such as methylene chloride or carbon tetrachloride for 5 hours to one day under heating in order to produce the compound of formula (1d). As the oxidant for this reaction, manganese oxide or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, preferably manganese oxide can be used. 
In the process variant (f) according to Reaction Scheme 7 above, the compound prepared in the process variant (d) or (e) wherein R3xe2x80x2 is ethoxycarbonyl is used as the starting material. The starting compound is reacted with lithium hydroxide, sodium hydroxide, lower alkylamine or lower alkenylamine to produce the compound of formula (1e) wherein R3xe2x80x3 is hydroxycarbonyl, lower alkylcarbamoyl or lower alkenylcarbamoyl. 
In the process variant (g) according to Reaction Scheme 8 above, the compound prepared in the process variant (d) or (e) wherein R3xe2x80x2 is ethoxycarbonyl is first reduced by using borane-methylsulfide complex(BMS complex) or sodium bis(2-methoxyethoxy)aluminumhydride for several hours under room temperature to warming to produce the compound of formula (12). The compound of formula (12) thus obtained is then reacted with aryl(or heteroaryl)(lower)alkyl halide or aryl(or heteroaryl)(lower)alkylmethylsulfonyloxy, for example, benzyl bromide or (2-pyridyl)methyl(methylsulfonyl)oxy in the presence of a base such as sodium hydride or potassium t-butoxide in a solvent such as tetrahydrofuran or dimethylformamide to produce the compound of formula (1f) wherein R3xe2x80x3xe2x80x2 is lower alkyl substituted by aryl(lower)alkoxy or 5- or 6-membered heteroaryl(lower)alkoxy containing nitrogen as the hetero atom. 
In the process variant (h) according to Reaction Scheme 9 above, 3xe2x80x2,4xe2x80x2-methylenedioxyacetophenone is reacted with bromine using a solvent such as carbon disulfide, chloroform, carbon tetrachloride to produce the 2-bromo compound of formula (13) which is then reacted with thiolacetic acid in the presence of a base such as triethylamine, pyridine or diisopropylethylamine under 0xc2x0 C. to slight warming to produce the desired 1-benzo[3,4-d]1,3-dioxolan-5-yl-2-acetylthioethan-1-one of formula (1g).
The compound of formula (1) of the present invention can be prepared by processes described above or by processes appropriately combined therefrom. Those processes will be more specifically explained in the following examples.
The hepatoprotective and therapeutic effects of the compound of formula(1) on liver diseases according to the present invention were determined through carbon tetrachloride model, D-galactosamine model, thioacetamide model and D-galactosamine/lipopolysaccharide model.
The carbon tetrachloride model(see, Philippe letteron et al., Biochemical Pharmacology, 39, 12, 2027-2034, 1990; Tips, 10, 1989; Kyoichi Kagawa et al., Japan J. Pharmacol., 42, 19-26, 1986; K. T. Liu and P. Lesca, Chem. Biol. Interactions, 41, 39-47, 1982; Richard O. et al., J. Biological Chemistry, 236, 2, 1961) is used most generally. This model has been established based on the internal phenomena that carbon tetrachloride is converted by cytochrome P-450 to a toxic free radical trichloromethyl(CCl3.), this radical is strongly bound to a thiol group of membrane protein on microsome of liver to form a lipid radical, which is then converted to a peroxy radical in the presence of oxygen to facilitate the peroxidation reaction of membrane lipid. That is, carbon tetrachloride suppresses protein biosynthesis in the liver, induces the increase of ALT and AST values in blood, and further causes histologically the centrilobular necrosis of the liver.
The hepatoprotective effect of the compound of formula (1) was also identified by D-galactosamine model(see, Koji Hase et al., Biol. Pharm. Bull., 20, 4, 381-385, 1997; Jun-ichi Nagakawa et al., J. Pharmacology and Experimental Therapeutics, 264, 1, 1992; Toxicology of the Liver, Raven Press, New York, 1985). The N-acyl galactosamine resulted from the excess administration of D-galactosamine acts as a substrate for UDP-galactosamine or UDP-N-acyl galactosamine. But, the inordinate synthesis of UDP-glucose and UDP-hexosamine may inhibit the binding of UTP in the liver cells and biosynthsis of UDP-glucose and UDP-galactose, which finally causes the structural and functional changes of the liver cell membranes. Consequently, it results in sporadic necrosis and the increase of ALT and AST values in blood. Since the toxicity caused by D-galactosamine looks similar to the symptoms of viral hepatitis, D-galactosamine model is also used conventionally as a model for viral hepatitis.
The hepatoprotective effect of the compound of formula (1) was also identified by thioacetamide model(see, Masuda Yasusuke and Nakayama Nobue, Biochemical Pharmacology, 31, 17, 2713-2725, 1982; Liu Jie et al., Acta Pharmacologica Sinica, 16, 2, 97-102, 1995; Story D. L. et al., J. Tox. Environ. Health, 11, 483-501, 1983; Gomez-Lechon M. J. et al., Xenobiotica, 18, 6, 725-735, 1988) which is frequently used as an experimental model for liver injuries. This model has been established based on the phenomena that thioacetamide activated by P-450 S-oxidation inordinately increases the influx of calcium ion(Ca++) into the liver cells, which in turn activates calcium ion-dependent phospholipase, protease and endonuclease, changes the cell frame through the calcium ion, exhausts ATP, and consequently destroys the liver cells. Thioacetamide also damages ureogenesis pathway in liver, and the metabolite of thioacetamide inhibits the transfer of RNA and makes the liver cell membranes unstable. A series of these activities results in the destruction of the liver cells, which in turn causes the increase of ALT and AST values in blood and also causes histologically the centrilobular necrosis.
Further, the D-galactosamine/lipopolysaccharide model(see, Chris Galanos et al., Proc. Natl. Acad. Sci. USA, 76, 11, 5939-5943, 1979; Koji Hase et al., Biol. Pharm. Bull., 20, 4, 381-385, 1997; Junichi Nagakawa et al., The Journal of Pharmacology and Experimental Therapeutics, 264, 1, 1992; Nolan J. D., Hepatology, 1, 458-465, 1981; Wendel A., Methods Enzymol., 186, 675-680, 1990) which is also used in the present invention for the identification of hepatoprotective effect is based on the phenomena that liver injuries are caused by immune responses in mouse. It has been reported that such kind of liver injuries are caused not by direct tissue lesions due to the chemicals but by secretion of tumor necrosis factor(TNF)-xcex1, reactive oxygen, etc.(see, Hishinuma I. et al., Hepatology, 12, 1187-1191, 1990, Hase K. et al., Phytother. Res., 10, 387-392, 1996). Here, D-galactosamine supresses protein biosynthesis and as well highly increases the sensitivity of the liver cells for lipopolysaccharide by reducing uridine nucleotide. That is, when a low dose of lipopolysaccharide which does not cause any toxicities by unitary administration is administered to a mouse together with D-galactosamine, amplification of toxicity for liver injuries may occur through an immunological pathway due to the secretions of TNF-xcex1 and reactive oxygen from macrophage in the liver, which in turn causes the increase of ALT and AST values in blood due to the loss of function and necrosis of the liver cells.
In the present invention, the compound of formula (1) was orally administered to rats and mice as experimental animals for once, and then the protective activity of the compound against liver injuries caused by carbon tetrachloride, D-galactosamine, thioacetamide or D-galactosamine/lipopolysaccharide was measured.
The degree of liver damage in the experimental animals was determined by measuring ALT and AST values in blood(see, Biol. Pharm. Bull., 20, 4, 381-385, 1997; Toxicology and Applied Pharmacology, 95, 1-11, 1988), and the hepatoprotective activity was calculated based on the following formula(see, Planta Medica, 55, 127-132, 1989). At this time, the hepatoprotective activity can also be calculated based on the same formula by replacing ALT value with AST value.             [              1        -                                                                                                  ALT value of the compound-treated group                                    -                                                                                                      ALT  value  of  the  normal  group                                                                                                                                              ALT  value  of  the  control  group                                    -                                                                                                      ALT  value  of  the  normal  group                                                                        ]        xc3x97    100    ,
in the above formula
the normal group means a group to which only a solvent is administered,
the control group means a group to which carbon tetrachloride, D-galactosamine, thioacetamide or D-galactosamine/lipopolysaccharide is administered, and thus the liver cells of the experimental animals are impaired, and
the compound-treated group means a group to which the compound of formula (1) according to the present invention is orally administered for once, and then one hour after the hepatotoxic material(carbon tetrachloride, D-galactosamine, thioacetamide or D-galactosamine/lipopolysaccharide) is administered.
The experimental results show that the compound of formula (1) of the present invention exhibits a superior hepatoprotective effect to silymarin or DDB well known as a hepatoprotective agent. Further, the present inventors have performed the acute toxicity test for the compound of formula (1) according to the present invention using mouse as the test animal, and therefrom it has been identified that the compound of the present invention is considerably safe since LD50 of the compound is more than 2,000 mg/kg when it is orally administered for once. Therefore, it is concluded that the compound of formula (1) according to the present invention is safe and also has an excellent hepatoprotective and therapeutic activity for liver diseases.
When the pharmaceutical composition according to the present invention is used for clinical purpose, it may be formulated into solid, semi-solid or liquid pharmaceutical preparations for oral or parenteral administration by combining the compound of formula (1) with a pharmaceutically acceptable inert carrier.
The pharmaceutically acceptable inert carrier which can be used for this purpose may be solid or liquid. It may be one or more selected from a group consisting of diluents, flavouring agents, solubilizing agents, lubricants, suspending agents, binders, swelling agents, etc. Specific example of the solid or liquid carrier which may be suitably used in the present invention includes lactose, starch, mannitol, cottonseed oil, etc.
When the active compound of formula (1) of the present invention is used as a medicine for prevention and treatment of liver diseases, it is preferably administered in a dose of 0.01 to 10 mg per kg of body weight per day at the first stage. However, the administration dosage can be varied with the requirement of the subject patient, severity of the liver diseases to be treated, the selected compound and the like. The prefered dosage suitable for a certain condition can be determined by a person skilled in this art according to a conventional manner. In general, the treatment is started from the amount less than the optimal dosage of the active compound and then the administration dosage increases little by little until the optimal therapeutic effect is obtained. As a matter of convenience, the total daily dosage may be divided into several portions and administered over several times.
The present invention will be more specifically explained by the following examples. However, it should be understood that the examples are intended to illustrate but not to in any manner limit the scope of the present invention.